For immunostaining analysis of the cellular localization of OTRs, HEK293T cells co-transfected with an HA-tagged OTR and mScamem (10:1 ratio) were fixed in PBS (FUJIFILM Wako) containing 4% (wt/vol) paraformaldehyde for 10 min at room temperature. Next, cells were permeabilized and blocked for 30 min at room temperature in blocking solution (PBS containing 0.2% TritonX-100 and 5% normal goat serum (FUJIFILM Wako)) and then incubated with an anti-HA antibody (rabbit; MBL; 1:2,000 dilution) for 30 min at room temperature. After washing with PBS containing 0.1% TritonX-100 (PBST), samples were incubated with Alexa 488-conjugated anti-rabbit IgG antibody (goat; Jackson ImmunoResearch) for 30 min at room temperature. After three washes with PBST, stained cells in PBS were analyzed by microscopy.
Phosphate buffered saline (pbs)
Manufactured by Fujifilm
Sourced in Japan, United States
PBS (Phosphate Buffered Saline) is a commonly used buffer solution in various laboratory applications. It is a balanced salt solution that maintains a stable pH and osmotic environment for various biological samples and experiments. The core function of PBS is to provide a physiologically compatible medium for the preservation and manipulation of cells, tissues, and other biological materials.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Fujifilm (5 mentions)
Fetal bovine serum (fbs), by Nichirei Biosciences (4 mentions)
Dimethyl sulfoxide (dmso), by Fujifilm (3 mentions)
Methanol, by Fujifilm (3 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (3 mentions)
Bz 9000, by Keyence (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Facscalibur, by BD (3 mentions)
Vacutainer cpttm tube, by BD (3 mentions)
Facsaria 3 ow cytometer, by BD (3 mentions)
Rpmi 1640, by Fujifilm (3 mentions)
Glutamine, by Fujifilm (3 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Facsaria 2, by BD (2 mentions)
Vacutainer cpt tube, by BD (2 mentions)
Collagenase type 1, by Fujifilm (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by Fujifilm (2 mentions)
Isopropanol, by Fujifilm (2 mentions)
Fv1000, by Olympus (2 mentions)
133 protocols using phosphate buffered saline (pbs)
1
Cellular Localization of Oxytocin Receptors
2
Quantitative Fluorescence Imaging of GFP, mRNA, and pDNA in Mouse Liver
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Mouse liver was perfused with phosphate buffered saline (PBS; Wako Pure Chemical Industries, Osaka, Japan) and 4% paraformaldehyde (PFA) in PBS (Wako). The tissue was then incubated with 4% PFA in PBS overnight, with 10% sucrose in PBS for 4 h, with 15% sucrose for 4 h, and 20% sucrose overnight at room temperature. The samples were frozen in optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan). Frozen sections were prepared at a thickness of 10 μm. The sections were treated with rabbit anti-GFP IgG (Invitrogen) at a 1:300 dilution for 2 h and then with Alexa 488 goat anti-rabbit IgG (Invitrogen) for 1 h at room temperature. Samples were mounted in ProLong Gold Antifade Reagent with DAPI (Invitrogen). Images were acquired using an LSM 510 confocal microscope (Carl Zeiss, Oberkochen, Germany) with a 20× objective at excitation wavelengths of 488 nm (Ar laser) for GFP, 633 nm (He-Ne laser) for Cy5, and 710 nm (MaiTai laser for 2-photon imaging) for DAPI. From these images, GFP expression and the cellular uptake of Cy5-labeled mRNA and pDNA into the cytoplasm and nucleus were evaluated using image analysis software (IN Cell Analyzer Workstation 3.7.1; GE Healthcare, Buckinghamshire, UK). The area of the nucleus was defined as the area stained with DAPI, and that of cytoplasm was defined as the area within 10 μm from the edge of the nucleus.
3
Visualizing TNFR1 and TNFR2 in ARPE-19 Cells
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ARPE-19 cells were cultured on glass bottom plates (AGC Techno Glass, Shizuoka, Japan) for 24 h, then co-cultured with MT2 and Jurkat cells using cell culture inserts (Thermo Fisher Scientific) for 48 h. After three washes with phosphate-buffered saline (PBS) (Wako Pure Chemical Corporation), ARPE-19 cells were fixed by cold fixing buffer (methanol/acetone, 1:1) at −20°C for 20 min and blocked with 10% FBS in PBS for 15 min. Cells were then incubated in the diluted primary antibodies for 1 h at room temperature, followed by incubation with Alexa fluor488-labeled anti-rabbit secondary antibody (Abcam, Tokyo, Japan) along with 4′,6-diamidino-2-phenylindole dihydrochloride (Cosmo Bio, Tokyo, Japan) incubation for 1 h at room temperature. The following antibodies were used as primary antibodies: TNF receptor 1 polyclonal antibody (Bioss Antibodies, Woburn, MA) and TNF receptor 2 polyclonal antibody (Proteintech, Chicago, IL). We scanned using a TCS-SP8 microscope (Leica Micro Systems, Wetzlar, Germany).
4
PBMC Immune Cell Sorting
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Primary PBMCs were stained with anti-CD3, anti-CD4, anti-CD7, and anti-CADM1 antibodies at room temperature in phosphate-buffered saline (−) (Wako) containing 2% fetal bovine serum and sorted into CD7+CADM1− cells and CADM1+ cells on a FACSAriaII (BD Biosciences). Flow cytometry data were analyzed using FlowJo software (Becton Dickinson & Company). The antibodies are listed in supplemental Methods .
5
Spheroid Formation Using Unique Method
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Spheroid formation using our unique method has been previously reported [16 (link),17 (link)]. Briefly, 1 µL of the cell suspension (6000 cells) was serially injected into the MC medium using an electronic micropipette (Eppendorf, Hamburg, Germany). Up to 100 spheroids were able to be formed in a 35 mm dish. After injection, the culture dish was incubated at 37 °C under 5% CO2 until spheroids formed in 2–3 days. To collect the spheroids from the MC medium, 0.1 g/mL cellulase (Onozuka RS; Yakult Pharmaceutical Industry, Tokyo, Japan) in phosphate-buffered saline (FUJIFILM Wako) was added onto the MC medium, and the culture dish was incubated for 60 min at 37 °C. This digestion step enabled the efficient collection of spheroids in a sample tube. Two conventional spheroid formation methods (hanging drop method and low-cell-attachment U-bottom plate method) were examined simultaneously for benchmark experiments. The cells were cultured for 2–3 days using each method. The number of cells per spheroid was determined considering that the spheroids should have a size that is easy to handle and that troubles such as internal necrosis should not occur.
6
Isolation and Characterization of PBMCs from Healthy Japanese Donors
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After written informed consent was obtained from 11 healthy Japanese adult donors in Wakayama Medical University (Table 1 ), peripheral blood was collected in a Vacutainer CPT tube (BD, USA). The tubes were centrifuged at 1,500×g at 25 °C for 20 min. The fraction containing the mononuclear cells was harvested and washed with cold phosphate-buffered saline (PBS) (Fujifilm Wako, Japan). Contaminated red blood cells were hemolysed using ammonium chloride solution (STEMCELL TECHNOLOGIES, Canada) at 25 °C for 10 min. After haemolysis, the cells were washed with cold PBS, counted, resuspended in the Roswell Park Memorial Institute-1640 medium (Sigma-Aldrich, USA) supplemented with 5% human AB serum (Sigma-Aldrich, USA), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Thermo Fisher Scientific, USA), and kept on ice until the start of the experiment (1 × 106 cells/890 μL). Due to the limited number of obtained PBMCs, peripheral blood was obtained repeatedly from part of donors, and used for each experiment. As frozen PBMCs did not produce IFN-α, only fresh PBMCs were used in this study.
Characteristics of PBMCs donors
| Age (years) | Total | Men | Women |
|---|---|---|---|
| 20–29 | 1 | 1 | 0 |
| 30–39 | 5 | 3 | 2 |
| 40–49 | 4 | 2 | 2 |
| 50–59 | 1 | 1 | 0 |
| Total | 11 | 7 | 4 |
| Mean age (years) | 39.1 | 37.7 | 41.5 |
7
Isolation and Cytotoxicity Assay Protocol
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Hydrochloric acid (HCl) (37%), sodium hydroxide (NaOH), chloroform (CHCl3), and ethanol (96%) were purchased from Merck (Darmstadt, Germany). Phosphate-buffered saline (PBS) and Collagenase type I were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Dulbecco’s modified eagle Medium-F12 were purchased from Sigma-Aldrich (Burlington, VT, USA). Pen-Strep solution and fetal bovine serum (FBS) were obtained from Gibco (New York, NY, USA).
8
Kidney Histology and Immunohistochemistry in PKD1 Pigs
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After PKD1 heterozygous mutant pigs and age-matched WT pigs were sacrificed under anesthetization, the kidney tissues were dissected, fixed in 4% paraformaldehyde in phosphate-buffered saline without calcium and magnesium (Wako Pure Chemical Industries, Osaka, Japan), embedded in paraffin, sectioned, and subjected to Masson’s trichrome staining. For immunohistochemical analysis, the fixed sections were treated with a mouse anti-PC1 monoclonal (clone: 7e12) antibody (1:200 dilution) overnight at 4 °C. After removing excess antibody, the sections were incubated with Histofine Simple Stain MAX PO (MULTI) (Nichirei Bioscience) and DAB chromogen for 30 min at 25 °C. The slides were counterstained with hematoxylin and visualized under a BIOREVO BZ9000 microscope (Keyence, Osaka, Japan).
9
Transparent Visualization of Fish Larvae
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Fish larvae were bleached and transparentized according to previous reports51 (link),52 (link), with several modifications. Formalin (10% formalin solution), potassium hydroxide (KOH), hydrogen peroxide, Triton-X, phosphate-buffered saline (PBS), HEPES, and glycerin were purchased from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). All other chemicals used in this study were of analytical grade. O. javanicus larvae (exposure group n = 6; negative control n = 2) were fixed in 10% formalin at 4 °C for 3 days. After removal of formalin, larvae were incubated in pre-bleaching solution (0.3% hydrogen peroxide in PBS) at room temperature overnight. Pre-bleaching solution was replaced with bleaching solution (3% hydrogen peroxide in PBS) and incubated at room temperature overnight. After bleaching, the bleaching solution was replaced with a tissue transparency solution (5% formalin, 5% Triton X-100, 1% KOH in PBS) and incubated at 42 °C for 24 h. In O. latipes, which has a less pigmented peritoneum than O. javanicus, bleaching was omitted, i.e., the tissue transparency solution was directly added after fixation, and incubated at 42 °C for 48 h. Transparency of each sample was confirmed under the microscope.
10
Mitochondrial Dysfunction and DNA Damage Assays
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5-ALA hydrochloride, RPMI 1680 medium, penicillin, streptomycin, fetal bovine serum (FBS), phosphate-buffered saline (PBS), NaOH, N,N-dimethylformamide, isopropanol, propidium iodide (PI), RNase, and D-luciferin were purchased from Wako Chemicals (Osaka, Japan). A modified Lowry protein assay kit was purchased from Pierce (Rockford, IL, USA). Aminophenyl fluorescein (APF) was purchased from Goryo Chemical (Sapporo, Japan). A DNA damage detection kit (containing γH2AX monoclonal antibody and secondary antibody) was purchased from Dojindo Laboratories (Kumamoto, Japan). A MitoPT® TMRE Mitochondrial Depolarization Assay Kit was purchased from ImmunoChemistry Technology (Bloomington, MN, USA).
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